Multifunctional bioactivity of Moringa oleifera seed acetone extract and its gold nanoparticle formulation: Immunomodulation, anticancer activity, antibacterial properties, acute toxicity, and oxidative stress assessment

1. Introduction

Medicinal and aromatic plants, particularly those utilized for their pharmacological property, have been a natural source of medicine and cure for numerous centuries (Chaachouay and Zidane, 2024). The fragrance of a few plants has captivated human senses always and aroused conventional methods for the extraction of essential oils from medicinal and aromatic plants. The therapeutic application of some species is traced back to ancient civilizations, which were mostly reserved for the elite who had botanical information (Chaachouay and Zidane, 2024; Santacroce et al., 2021). In recent years, there has been significant attention devoted to natural remedies since mainstream medicine provides a holistic approach to enhancing patient well-being and presents traditional knowledge of the beneficial nature of plants, which is helpful in the production of dietary supplements and medicines (Marques et al., 2021).

Moringa oleifera is a tropical tree that heightens the interest of scientists and researchers due to its high nutritional value and medicinal properties. M. oleifera is derived from India, tropical nations, and Africa and possesses incredible nutritional and medicinal properties. M. oleifera possesses a vast array of pharmacological activities because it is rich in numerous bioactive compounds, mostly in the leaves, seeds, flowers, roots, and pods (Ercan et al., 2021). Almost all the components of the M. oleifera tree are utilized in food and medicine. It has been hypothesized that the bioavailability of phytochemicals such as flavonoids, alkaloids, tannins, and glycosides can be credited for the palatable and otherwise curative nature of Moringa. Seed extracts of M. oleifera are routinely being used in the practice of traditional medicine (Meireles et al., 2020). “Drumstick seeds” or “moringa pods” refer to the seeds of Moringa and have conventionally been utilized to treat many diseases in Asia and Africa, including hypertension, diabetes, and gastritis.

Moringa seeds possess intensive antioxidant activity, which could be attributed to the flavonoids, tannins, and ascorbic acid present in them. Moringa seeds, oil, and extracts have been reported to possess remarkable anti-inflammatory activities. Moringa seed extracts are non-toxic and biocompatible and exhibit minimal animal toxicity and irritation to the eyes and skin (Liu et al., 2022). Moringa oleifera seeds possess several bioactivities, and among them, their antibacterial activity (Chandrashekar et al., 2020; Dzuvor et al., 2022). Moringa leaves have also been found to be of nutritional and medicinal importance, and its seeds are used to purify water. Seeds have about 36–42% oil that is made up of more than 80% mono/polyunsaturated fatty acids, with considerable levels of oleic and linoleic acids, which both have a biosystem-compatible ω-9 and ω-6 polyunsaturated fatty acid (PUFA) chain (Fejér et al., 2019).

However, vegetative research has established that seeds are likely to be made up of a greater proportion of bioactive compounds compared to the rest of the plant. Literature on the chemical structure and biological properties of Moringa oleifera seeds seems to be lacking, especially when compared to other studies on leaves. Maceration extracts of Moringa oleifera seeds are potent microbial growth inhibitors, and Moringa seeds are antifungal against several human pathogenic fungi in vitro. Extracts combined of seeds and leaves were conventionally applied in managing diabetes (Tekwani et al., 2022). Studies on the plant material of Moringa oleifera and the antioxidant activity of leaf extracts of this plant have received a lot of attention. Minimal literature can be found on the chemical constitution and bioactivities, other than antifungal or antimicrobial, of seed extract. It has been shown that Moringa extracts exert in vitro activities against the human immune system; however, only Moringa oleifera dried leaf extracts were investigated.

Immune cell activities have already been assayed using a wide variety of naturally occurring products. Although some of these natural bioactive products stimulate the immune response and are proinflammatory, others are known to be immunosuppressive or anti-inflammatory. Except for artemisinin, it has been shown that traditionally used medicinal plants of clinical significance can only act as immunomodulators when higher doses of plant-derived extracts are ingested (Alanazi et al., 2023; Alhazmi et al., 2021). Therefore, understanding hemp’s intrinsic bioactivity as an immunomodulatory agent may be invaluable in the development of novel natural product-derived drugs. Nanoparticles (NPs) have a diameter of 100 nm or less. Certain unreliable precursors help stabilize unstable particles that wouldn’t exist otherwise. Most commercially available NPs are metastable, with only a small fraction in thermodynamic equilibrium. Their size affects the properties of bulk materials and can lead to unexpected changes, necessitating special classification. For instance, NP color varies with incidence direction and thickness, rather than being dichromatic (Joudeh and Linke, 2022).

Nanotechnology is expected to revolutionize drug discovery, biomaterials, and electronics by providing innovative tools for designing and synthesizing new nanomaterials. It has emerged as a promising frontier technology, enabling the reduction of materials to ultrafine sizes. NPs, which can be made in various shapes and sizes, have diverse applications, including catalysis, antimicrobial activities, environmental remediation, therapeutic drugs, and self-cleaning surfaces. Understanding the principles and characteristics of NPs is important for advancing numerous technologies (Malik et al., 2023). NPs are produced using a range of effective methods that are applicable in industrial and high-technology products. Metal NPs are formed by subjecting particles with metal to a high-energy environment. The techniques of fabrication are also classified into two main categories: top-down and bottom-up synthesis methods. Top-down is the process of downsizing bulk material to NPs, and bottom-up is the construction of NPs from atomic or molecular building blocks. Major synthesis methods are evaporation-condensation, plasma synthesis, attrition, liquid processing, and solid-phase and gas-phase chemical reduction (Pandit et al., 2022; Yaqoob et al., 2020). Biosynthesis of NPs is a new field of nanotechnology, and the phytosynthesis pathway has a quick, available, covering, and harmless method of phytoproduct reduction. The manufacturing procedure of such NPs is environmentally friendly and appropriate for industry because it lessens the consumption of energy and gives more eco-friendly correctory products. Silver NPs (AgNPs) are the most used NPs because they are extensively used in industry, health, textiles, biomedical electronics, and so biology (Tiwari et al., 2021).

Gold NPs (AuNPs) have attracted considerable interest within the realm of biomedical applications due to their unique physicochemical properties. These factors encompass a significant surface area-to-volume ratio, the ability to customize surface chemistry, and outstanding optical characteristics, especially demonstrated by surface plasmon resonance (SPR). AuNPs have garnered significant attention in the field of biomedical applications owing to their distinctive physicochemical characteristics. These include an extensive surface area-to-volume ratio, customizable surface chemistry, and exceptional optical properties, particularly exemplified by SPR. Such features make AuNPs highly suitable in diagnostics, drug delivery, imaging, and treatment (Dreaden et al., 2012). AuNPs can be chemically, physically, or biologically synthesized. Citrate or borohydride reduction by chemicals is common, but the greener choice offered by the plant extracts or microbial method for synthesis is advantageous (Thakur et al., 2012). Biomolecule surface modification (e.g., with antibodies, peptides, or DNA) enhances their biocompatibility and targeting capacity (Seidu et al., 2022). AuNPs are good carriers of drugs, genes, and proteins due to their ability to be conjugated with various biomolecules. Target-specific release and controlled drug delivery by them reduce systemic toxicity (Dykman and Khlebtsov, 2017). AuNPs enhance the sensitivity of biosensors for the identification of biomolecules (e.g., DNA, proteins) by colorimetric assays or surface-enhanced Raman spectroscopy (SERS) (Rosi and Mirkin, 2005). AuNPs are used in photothermal therapy, where absorption of near-infrared (NIR) light induces local hyperthermia, selectively destroying cancer cells (Hao et al., 2014).

Because of their strong X-ray absorbance, AuNPs are utilized as contrast agents when computed tomography imaging is employed (Das et al., 2023). While generally biocompatible, AuNP toxicity is size-, shape-, and surface-coating-dependent. Smaller particles (<5 nm) are more likely to be cytotoxic as they are internalized by cells and induce oxidative stress (Alkilany and Murphy, 2010).

Moringa oleifera seed acetone extract’s anticancer efficacy against rabidly growing tumor cells (HT-29 col cell line) and fast-dividing normal cells (proliferation-activated splenic cells) was examined in the current work. Apoptosis, cell cycle, oxidant/antioxidant properties, NP production and characterization, antibacterial, acute toxicity, and cell cytotoxicity were among the additional biological capabilities of MSAE that were investigated.

2. Materials and Methods

3. Statistical Analysis

The analysis of the data was performed utilizing a two-way ANOVA to assess the statistical significance among the groups, with all experiments being executed in triplicate. The statistical evaluation was carried out using GraphPad Prism software (GraphPad Prism Version 5). A threshold for statistical significance was set at p < 0.05, and the findings are reported as the mean ± standard deviation (SD).

4. Results

4.1 AuNPs production

The formation of AuNPs was monitored by assessing the color change in the tetrachloro-auric acid (HAuCl₄) and MSAE mixture. After the color transition (Fig. 1), the synthesis of AuNPs was evaluated using spectrophotometric methods (Fig. 1). The outcomes indicated the existence of a specific absorbance peak associated with AuNPs within the wavelength spectrum of 450-530 nm.

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Fig. 1.

Synthesis of AuNPs utilizing the acetone extract of Moringa oleifera seed; (a) MSAE; (b) light absorbance profile of the MSAE; (c) MSAE after the incorporation of gold chloride; (d) light absorbance profile of MSAE combined with AuNPs.

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4.2 Functional groups

The FTIR spectrum of the sample was acquired with eight sample scans and eight background scans. The transmittance peaks observed in the spectrum have been listed in Table 1, along with their corresponding wavenumbers and transmittance values.

Table 1. List of wavenumber (cm^⁻1) and possible functional group/bond found in Moringa oleifera seed acetone extract.

Wavenumber (cm⁻1)	Transmittance (%)	Possible functional group/bond
3377.4	94.94	O-H stretch (alcohols, water)
2981.4	93.183	C-H stretch (alkanes)
2853.3	93.458	C-H stretch (alkanes)
2924.1	88.969	C-H stretch (alkanes)
1696.6	89.053	C=O stretch (carbonyl)
1686.6	86.053	C=O stretch (carbonyl)
1747.1	95.507	C=O stretch (carbonyl)
1244.2	93.125	C-O stretch (ethers, esters)
1169.2	94.137	C-O stretch (ethers, esters)
684.1	93.324	C-H bend (aromatics)
693.8	93.477	C-H bend (aromatics)
562.8	92.529	C-Br stretch (alkyl halides)
546.1	92.009	C-Br stretch (alkyl halides

The FTIR spectrum (Fig. 2) is plotted as transmittance (%) versus wavenumber (cm^⁻1) over the range of 4000–400 cm^⁻1. The spectrum shows characteristic absorption bands corresponding to functional groups such as O-H, C-H, C=O, and C-O bonds. O-H Stretch (3377 cm^⁻1) suggests the presence of alcohols or water in the sample. C-H Stretches (2981–2853 cm^⁻1) indicate aliphatic hydrocarbon chains. C=O Stretches (1696–1747 cm^⁻1) likely due to carbonyl groups in aldehydes, ketones, or esters. C-O Stretches (1244–1169 cm^⁻1) consistent with ethers, esters, or alcohols. Peaks below 1000 cm^⁻1 may indicate aromatic C-H bending or halogen-containing compounds (e.g., C-Br).

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Fig. 2.

FTIR spectra of Moringa oleifera seed acetone extract.

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4.4 Levels of sugar and protein

The findings from the HPLC analysis indicated that MSAE contains a negligible quantity of sucrose and no detectable levels of fructose, glucose, or maltose (Fig. 3). Additionally, SDS-PAGE analysis demonstrated that no protein fractions were detectable.

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Fig. 3.

A representative HPLC chromatogram of MSAE illustrating the concentrations of fructose, glucose, sucrose, and maltose.

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4.10 Cell cycle assessment

To determine whether the observed suppressive power of the extracts on the colon cancer cells was attributable to the induction of the arrest of the cell cycle, the human colon cancer HT-29 cell line was subjected to treatment with MSAE at concentrations equivalent to IC₅₀ for a duration of 48 h. The progression of the cell cycle was evaluated using flow cytometry. Following the 48-h treatment with MSAE and MSAE+AuNPs, there was a statistically significant increase (p>0.001) in the cell population within the G2/M phase, rising from 26.64% in the untreated control to 48.35%. This escalation in the G2/M population was associated with a concomitant reduction in the cell population within the G1 phase, which decreased from 46.93% in the untreated control to 26.93% in the MSAE-treated group, with a recorded value of 41.66% (Fig. 4).

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Fig. 4.

Progression of the cell cycle. (a) Untreated colon cancer cells; (b) Colon cancer cells treated with MSAE. (c) The distribution and proportion of cells within the preG, G1, G2/M, and S phases of the cell cycle.

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4.11 Apoptotic effects of MSAE

The apoptotic effects of MSAE were assessed using Annexin V staining (Fig. 5). The MSAE induced apoptosis in the cells, resulting in a significant (p<0.05) increase in the number of apoptotic cells in the extract-treated group (21.39%) compared to the untreated group (2.25%). The elevation in the percentage of apoptotic cells subjected to MSAE at the early stage (9.41%) was noted to be non-significantly lower than that observed at the late stage (10.35%). Furthermore, at the late stage, MSAE-treated cells (10.35%) exhibited a higher percentage of apoptosis than the untreated cells (0.44%). The observed reduction in cellular proliferation is ascribed to apoptosis as opposed to necrosis, as the percentage of necrotic cells in the MSAE-treated group was recorded at 1.63% across both early and late phases.

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Fig. 5.

Colon cancer cells (HT-29) staining with Propidium iodide/anti-Annexin V for the measurement of the percentages of apoptotic cells. (a) Depicts the untreated cells; (b) Illustrates the cells treated with MSAE; (c) A comparison of the apoptotic cells observed in both the untreated and MSAE-treated populations.

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5. Discussion

The research presented an extensive examination of the production, characterization, and bioactivity of AuNPs derived from MSAE. The results underscore the promising applicability of MSAE and MSAE-AuNPs in antimicrobial, anticancer, and immunomodulatory fields, supported by thorough experimental data.

Confirmation of AuNP synthesis was achieved through the observation of a distinctive color change and a specific absorbance peak within the 450–530 nm spectrum, corresponding with the SPR characteristic of AuNPs (Daniel and Astruc, 2004). The SPR peak confirms NP synthesis, consistent with prior studies using plant extracts as reducing and stabilizing agents (Mittal et al., 2013). The phytochemicals present in MSAE, including polyphenols and flavonoids, likely facilitated the reduction of gold ions (Au³⁺) to Au⁰, as these compounds are recognized for their redox properties (Zuhrotun et al., 2023).

FTIR analysis indicated the existence of active groups such as O-H, C-H, and C=O, suggestive of alcohols, alkanes, and carbonyl compounds, which are typically associated with bioactive substances like flavonoids, terpenoids, and alkaloids known for their roles in NP synthesis and stabilization (Kumar and Yadav, 2009). Additionally, the presence of C-Br stretches implies halogenated molecules that may augment the bioactivity of the extract (Tella and Oseni, 2018).

SEM analysis showed that the AuNPs were spherical in form. The uniformity in shape and size is crucial for biomedical applications, as it ensures consistent behavior in biological systems (Daniel and Astruc, 2004). The slight polydispersity observed could be attributed to the natural variability in the extract’s composition, a common challenge in green synthesis (Iravani et al., 2014).

The absence of detectable sugars (except trace sucrose) and proteins in MSAE, as confirmed by HPLC and SDS-PAGE, suggests that the bioactivity of the extract is primarily due to non-proteinaceous and non-carbohydrate compounds, such as polyphenols or terpenoids (Yogi et al., 2016). This aligns with studies emphasizing the role of secondary metabolites in plant-mediated NP synthesis (Marslin et al., 2018).

The present study quantified the ROS content in MSAE. The outcomes revealed a substantial ROS presence within MSAE. The purpose of quantifying ROS content was to identify external factors that could influence other parameters under investigation in this study, such as apoptosis. A slight escalation in ROS levels in MSAE-treated cells as opposed to controls suggests a mild induction of oxidative stress, which may play a role in its antimicrobial and anticancer activities (Nogueira and Hay, 2013). Modulation of ROS is recognized as a mechanism of action for numerous therapeutics derived from plants (Saad Entsar et al., 2013).

MSAE-AuNPs demonstrated marked antimicrobial effectiveness, particularly against Bacillus subtilis. The increased efficacy relative to MSAE alone highlights the synergistic effect of AuNPs, likely attributable to their high surface area and their capacity to disrupt microbial membranes (Sa’ed et al., 2024). The diminished activity noted against E. coli may reflect the intrinsic resistance of Gram-negative bacteria, which is typically linked to their outer membrane structures (Breijyeh et al., 2020). As anticipated, ciprofloxacin displayed superior efficacy, serving as a positive control (Sharma et al., 2010).

AuNPs have garnered significant interest in recent years, given their exceptional physicochemical properties and potential applications in biomedicine, particularly regarding antibacterial functions (Dykman and Khlebtsov, 2011). In contrast to bulk gold, which remains inert, nano-sized gold presents enhanced surface reactivity, allowing for effective interaction with bacterial cells. Numerous investigations indicate that AuNPs elicit antibacterial actions through various mechanisms. Positively charged AuNPs can bind to the negatively charged bacterial cell membrane, resulting in membrane disruption and the leakage of intracellular contents (Li et al., 2014). AuNPs may also initiate oxidative stress within bacteria by producing ROS, thereby damaging lipids, proteins, and DNA (Garcia-Pardo et al., 2022). Furthermore, AuNPs can interfere with bacterial metabolic functions by binding to crucial enzymes and inhibiting their activity (Mutalik et al., 2023). Nonetheless, challenges persist in optimizing parameters such as size, shape, and surface modifications to enhance antibacterial efficiency (Menichetti et al., 2023). Further in vivo investigations are essential to evaluate the long-term biocompatibility and therapeutic capacity of these NPs.

MSAE and MSAE-AuNPs exhibited cytotoxicity in a manner that is dependent on the dosage, with IC₅₀ values of 981.24 μg/mL and an unreached level for MSAE-AuNPs, respectively. The greater effectiveness of MSAE-AuNPs suggests that the NPs improve the delivery of bioactive constituents (Nisha et al., 2024). The stimulation observed at reduced concentrations (e.g., 100.79% at 250 μg/mL) may imply the phenomenon of hormesis, which presents a biphasic response whereby low doses promote cellular growth (Thong and Maibach, 2008).

AuNPs are recognized as promising anticancer agents owing to their distinctive physicochemical characteristics, biocompatibility, and ability to enhance drug delivery as well as photothermal therapy. Several studies have highlighted their potential for cancer diagnosis and treatment through various mechanisms, including targeted drug delivery, induction of apoptosis, and enhancement of radiotherapy. AuNPs can be functionalized with chemotherapeutic agents, thereby improving solubility and enabling specific delivery to tumor tissues. For example, paclitaxel-loaded AuNPs showed improved cytotoxicity in MCF-7 cells compared to free paclitaxel (Patra et al., 2008). The induced permeability and retention phenomenon allows AuNPs to preferentially accumulate in tumor areas, thereby minimizing systemic toxicity. Furthermore, AuNPs, specifically gold nanorods and nanoshells, exhibit strong absorption in the NIR spectrum, facilitating targeted tumor ablation. Huang et al., (2006) demonstrated that gold nanorods conjugated with anti-EGFR antibodies successfully targeted and annihilated oral cancer cells upon NIR laser exposure, while preserving healthy tissue. AuNPs are reported to amplify the effects of radiotherapy by increasing localized radiation absorption due to their high atomic number. Hainfeld et al., (2004) noted that tumor-bearing mice treated with AuNPs alongside X-ray radiation displayed significantly elevated survival rates in comparison to radiation therapy alone. Research indicates that AuNPs can foster the production of ROS, thus precipitating apoptosis in cancer cells. Pan et al., (2009) observed that citrate-capped AuNPs generated oxidative stress in human leukemia cells (K562), resulting in DNA damage and eventual K562 cell death.

The immunostimulatory properties of MSAE (2071% growth stimulation at 1000 μg/mL) emphasize its potential as an immunomodulatory agent. This aligns with studies on This observation corresponds with studies that have reported Moringa oleifera’s capacity to enhance immune system responses (Ibrahim et al., 2022b).

The lack of effect noted in the acetone control verifies the bioactivity present in the extract. Numerous investigations have analyzed the impact of AuNPs on immune cell functionality, including splenic cells that are essential for immune responses. Prior studies have confirmed that AuNPs can modulate immune cell activities contingent upon their size, shape, surface charge, and concentration. For instance, Chen et al., (2009) reported that citrate-coated AuNPs (20 nm) were uptaken by splenic macrophages without inducing significant cytotoxic effects, thereby indicating good biocompatibility at reduced concentrations. Similarly, Zhang et al., (2011) found that smaller AuNPs (5–10 nm) were absorbed more effectively by splenocytes compared to larger entities (50 nm); however, excessive accumulation could incite oxidative stress and mild inflammation. Conversely, Sumbayev et al., (2013) concluded that PEGylated AuNPs diminished pro-inflammatory cytokine production in murine splenocytes, revealing potential anti-inflammatory properties. Nonetheless, elevated doses of AuNPs were observed to inhibit lymphocyte proliferation as reported by Babin et al., (2013), indicating a dose-dependent immunomodulatory response. Our findings corroborate these conclusions, demonstrating that nano-gold at optimal concentrations does not provoke significant cytotoxicity in normal rat splenic cells, yet may influence immune function.

The observed upregulation of p53 (3.67-fold with MSAE, 7.61-fold with MSAE-AuNPs) suggests activation of pathways associated with apoptosis and cell cycle arrest, consistent with the tumor-suppressive functions of p53 (Zilfou and Lowe, 2009). The augmented fold increase with AuNPs implies enhanced cellular uptake or stability of the bioactive substances (Alkilany and Murphy, 2010).

Previous studies have established that Moringa oleifera seed extract exhibits cytotoxic effects across various cancer cell lines. A report by Al-Asmari et al., (2015) revealed that the seed extract enhanced programmed cell death in cells of colorectal cancer, coinciding with elevated p53 levels, thus highlighting its role in promoting apoptosis. Similarly, Tiloke et al., (2016) noted that Moringa oleifera leaf and seed extracts elevated p53 expression in lung cancer cells, pushing cell cycle arrest at the G2/M phase. The mechanism underlying p53 activation via Moringa oleifera may involve pathways related to oxidative stress and DNA damage response. Ramli et al., (2024) suggested that bioactive constituents such as glucosinolates and flavonoids present in the seed extract could stabilize p53 by inhibiting its MDM2-mediated degradation. Furthermore, a study by Rajkumar et al., (2024) demonstrated that niazimicin, a bioactive component found in Moringa seeds, enhanced p53 transcriptional activity, resulting in elevated expression of downstream pro-apoptotic factors such as BAX and PUMA. Collectively, these findings suggest that Moringa oleifera seed extract is capable of upregulating p53, thereby contributing to its anticancer properties. Nevertheless, further research is warranted to elucidate the exact molecular interactions and prospective clinical applications.

The notable rise in G2/M phase cells (48.35% vs. 26.64% control) indicates cell cycle arrest, a characteristic mechanism of action for anticancer agents (Schwartz and Shah, 2005). The observed decrease in G1 phase cells further corroborates this, as G2/M phase arrest inhibits entry into mitosis (Lang et al., 2024). The capability of MSAE to induce cell cycle arrest while altering DNA content has been documented in various studies, affirming its potential as an anticancer entity. Research has indicated that MSAE triggers cytotoxic outcomes by disturbing cell cycle progression and promoting apoptosis across several cancer cell types. For example, a study done by Al-Asmari et al., (2015) evidenced that Moringa oleifera leaf extract induced G2/M phase arrest in human colorectal cancer cells (HCT-8), accompanied by a notable reduction in both cyclin B1 and cyclin-dependent kinase 1 (CDK1) expression, key regulators of the G2/M transition. Subsequent flow cytometric evaluations of DNA content indicated that MSAE exposure elevated the sub-G1 cell population, signifying apoptotic DNA fragmentation (Sreelatha et al., 2011). Another investigation by Berkovich et al., (2013) ascertained that Moringa oleifera extract produced dose-dependent DNA damage in pancreatic cancer cells (PANC-1), demonstrated through a comet assay, further supporting its genotoxic implications on malignant cells.

These collective findings imply that MSAE modulates checkpoints of the cell cycle and influences DNA integrity, potentially via the regulation of cyclins, CDKs, and tumor suppressor proteins.

The Annexin V assay confirmed the induction of apoptosis, with minimal necrotic occurrence. This selective induction of apoptosis is advantageous in oncological therapies as it mitigates collateral tissue damage (Elmore, 2007). The higher percentage of late-stage apoptosis (10.35%) suggests prolonged cytotoxic effects (Maiuri et al., 2009).

The promotion of apoptosis constitutes a fundamentally critical mechanism by which natural products exert anticancer and chemopreventive effects. Numerous investigations have substantiated that MSAE demonstrates significant apoptotic activity in various cancer cell lines, indicating its potential as an effective antitumor agent. Previous research has elucidated that MSAE facilitates apoptosis through multiple pathways, encompassing the activation of caspase cascades, regulatory modulation of Bcl-2 family proteins, and induction of ROS. Al-Asmari et al., (2015) indicated that Moringa oleifera seed extract application in human hepatocellular carcinoma (HepG2) cells resulted in increased activities of caspase-3 and -9, alongside the downregulation of anti-apoptotic Bcl-2 and the upregulation of pro-apoptotic Bax, signifying mitochondrial-mediated apoptotic pathways (Al-Asmari et al., 2015). In parallel, Guon and Chung, (2017) observed that Moringa oleifera seed extract initiated apoptosis in MCF-7 cells via ROS-dependent mechanisms, resulting in DNA fragmentation and cell cycle arrest at the G2/M phase (Guon and Chung, 2017). Furthermore, research conducted by Sreelatha et al., (2011) has illustrated that bioactive compounds derived from Moringa seeds, including glucosinolates and phenolic acids, contribute to its pro-apoptotic effects by suppressing NF-κB signaling pathways, thereby inhibiting cancer cell survival pathways (Sreelatha et al., 2011). These results are in alignment with more recent work by Rajkumar et al., (2024), which documented that MSAE augmented p53 expression in colon cancer cells, facilitating apoptosis and inhibiting cell proliferation (Rajkumar et al., 2024).

The normal ranges of hepatic and renal markers (ALT, AST, urea, creatinine) indicate low systemic toxicity, an essential attribute for therapeutic agents (Borlak et al., 2014). The increased levels of TBARS and decreased SOD/GSH in tissues treated with MSAE indicate the induction of oxidative stress, potentially mediating its bioactivity (Jomova et al., 2023). The tumor marker data (e.g., Arginase) necessitate further scrutiny to elucidate specific anticancer mechanisms. The assessment of liver and kidney function indicators, such as ALT, AST, urea, and creatinine, is imperative in determining the hepatoprotective and nephroprotective effects presented by natural compounds, including Moringa oleifera seed extract. Numerous studies have investigated the impact of Moringa oleifera on these biochemical parameters, affirming its therapeutic potential. Elevated ALT and AST levels frequently signify hepatic injury, typically instigated by oxidative stress, toxic substances, or inflammation. Research has indicated that Moringa oleifera seed extract can significantly modulate ALT and AST levels in animal models experiencing hepatotoxicity. For example, Al-Sultan et al., (2024) reported that pre-treatment with Moringa oleifera seed extract in paracetamol-induced liver damage in rats resulted in notably lower ALT and AST levels, indicating hepatoprotective properties attributable to its antioxidant constituents, such as flavonoids and phenolic compounds. Likewise, Adedapo et al., (2009) found that Moringa oleifera seed extract mitigated carbon tetrachloride (CCl₄)-induced hepatic damage by diminishing oxidative stress and normalizing liver enzyme levels.

Urea and creatinine serve as critical indicators of renal function, with elevations often reflecting renal impairment. Research suggests that Moringa oleifera seed extract may exert nephroprotective effects by alleviating these markers. A study by Al-Malki and El Rabey (2015) revealed that diabetic rats administered Moringa oleifera seed extract exhibited significant decreases in serum urea and creatinine concentrations, likely attributed to its hypoglycemic and antioxidant properties. Furthermore, Rana et al., (2016) noted that the extract alleviated nephrotoxic effects induced by cisplatin by lowering creatinine and urea levels, possibly through enhancement of renal antioxidant defenses. The beneficial impacts of Moringa oleifera seed extract on liver and kidney function markers can be ascribed to its rich phytochemical profile, encompassing polyphenols, flavonoids, and glucosinolates, which possess antioxidant, anti-inflammatory, and detoxifying properties (Leone et al., 2015). These compounds are instrumental in ameliorating oxidative injury, enhancing cellular repair mechanisms, and promoting the overall function of organs.

6. Conclusions

The research highlights the green synthesis of AuNPs utilizing MSAE, emphasizing their potential in antimicrobial, anticancer, and immunomodulatory applications. AuNPs were synthesized, confirmed by SPR results from 450 to 530 nm, and characterized using FTIR analysis, identifying functional groups (O-H, C-H, C=O) from bioactive compounds like polyphenols and flavonoids. SEM analysis revealed that AuNPs were spherical and uniform, with some polydispersity due to extract variability. Both MSAE and MSAE-AuNPs displayed dose-dependent cytotoxicity, enhancing bioactive compound delivery. Notably, antimicrobial effects were strong against Bacillus subtilis, while E. coli showed resistance due to the membrane structure. Anticancer effects included a notable upregulation of p53 (7.61-fold with AuNPs), G2/M phase cell cycle arrest (48.35%), and late-stage apoptosis induction (10.35%), linked to oxidative stress and mitochondrial pathways. MSAE exhibited immunostimulatory effects without significant cytotoxicity on normal cells. Hepatic and renal marker analysis in normal subjects showed minimal systemic toxicity, with elevated TBARS and reduced SOD/GSH indicating oxidative stress as a bioactivity mediator.